Several studies have generated RPE-like cells from human iPS cells either for the purpose of disease modeling or for pre-clinical animal testing. These RPE-like cells are often recognized by their pigmented and polygonal nature and their functional characterization is done only using assays like photoreceptor outer segments phagocytosis. Currently, there is no effort done to provide an operational definition of RPE cells generated in vitro from human iPS cells. To authenticate RPE generated in vitro from human iPS cells and to obtain an operational definition of these RPE cells, we have performed a comprehensive gene expression, structural, and functional analysis of 19 different iPS cell clones generated from genetically and epigenetically distinct tissue sources (e.g. fetal or adult cornea or RPE from the same donor eye). Our gene expression analysis included 154 RPE signature genes, 76 fetal RPE-specific genes, and 96 adult RPE-specific genes, miRNAs enriched in human fetal RPE (e.g. miR-184, 187, 200a, 200b, 204, 211, 221, 222), retina (e.g. miR-124a, 124b, 135b, 182, 183, 96), and pluripotent stem cells (e.g. miR-302 family). Our structural analysis included immunostaining for key RPE markers and transmission electron microscopy. In addition, we performed following functional assays on these RPE cells: ability to phagocytose photoreceptor outer segments; transepithelial resistance/TER and potential/TEP; apical membrane hyperpolarization due to reduced K+ concentration in the apical bath, mimicking in vivo light onset; reduction in intracellular pH induced by CO2 changes selectively in the apical bath; ability to regulate intracellular calcium, and transport water from the apical to basal side in response to the activation of purinergic P2Y2 receptors at the apical membrane. Our results show that irrespective of the tissue source, there is no inherent epigenetic bias in iPS cells that significantly affects the quality of RPE cells. The quality of iPS cell line and the donor allelic variability are the two major contributors to variability in the performance of pure-cultures of RPE derived from iPS cells. This study will help provide a detailed set of assays that can be used to functionally authenticate iPSC-RPE and provide data on functional variation seen in RPE derived from multiple different iPS cell lines originating from with distinct genetic and epigenetic tissue sources. Work for this project has been completed and has been submitted